The present invention relates to new low molecular weight protein fragments that are degradation products of the cartilage oligomeric matrix protein (COMP) unique antibodies to these fragments and methods of using such antibodies to measure the severity of arthritic conditions.
Cartilage Oligomeric Matrix Protein (COMP) is a pentamer of molecular weight 435,000 that is part of the extracellular matrix of cartilage. Each monomer of about 87,000 is synthesized and secreted by cartilage chondrocytes. COMP is thought to constitute about 1 percent of the wet weight of cartilage. COMP is similar in structure to the members of the thrombospondin gene family. They are similar in the type 3 repeats and the C-terminal region. Thrombospondins, although similar in structure to COMP, have activities to regulate cell migration, growth and proliferation (such as vascular smooth muscle) and inhibit the growth of endothelial cells (Newton et al., (1994) Genomics 24: 435-439).
The physiological function of COMP is not known but its presence in the synovial fluid and serum has been correlated with osteoarthritis (Sharif et al., (1995) 34: 306-310) and rheumatoid arthritis (Forslind et al., Brit. J. Rheumatology, (1992) 31: 593-598). It has also been proposed as a marker of cartilage turnover in synovial fluid and blood (Saxne and Heinegard, D. (1992) Br. J. Rheumatology 31: 583-591). Moreover, the synovial fluids of rheumatoid arthritis patients that have high levels of COMP contain a 65 kilodalton fragment and possibly (under reducing conditions) traces of a lower molecular weight species (Saxne and Heinegard, D. (1992) Br. J. Rheumatology 31: 583-591). A recent study also demonstrated that the cartilage as well as the synovial fluid from patients with osteoarthritis and rheumatoid arthritis contain fragments ranging from 43 kDa, 67-94 kDa, 150-200 kDa (Dicesare P. E. et al., (1996) J. Orthopaedic Res. 14: 946-955).
Another cartilage matrix protein, cartilage matrix glycoprotein (CMGP), has been found in the serum of osteoarthritic dogs (Fife and Brandt (1989) J. Clin. Invest. 84: 1432-1439) but it has not been shown to be the result of cartilage breakdown. U.S. Pat. No. 4,778,768 discloses the correlation of cartilage damage and measurement of proteoglycan and fragments. The release of the G1 domain of proteoglycan increased with disease severity in rheumatoid arthritis but the larger region of glycosaminoglycan-rich region (CS/KS domain) decreased under the same conditions (Saxne and Heinegard, (1993) Arthritis Rheum. 35: 385-390). This demonstrates that it has until now been unpredictable whether fragments of a protein can predict a disease state.
Aggrecan is a proteoglycan residing in the cartilage. This protein consists of keratan sulfate (KS) side chains. In arthritis, aggrecan degradation products have been found in the synovial fluids of patients. One way of measuring aggrecan degradation products in the synovial fluid and serum is to quantify by an ELISA using antibodies that recognize keratan sulfate side chains (Thonar E J-M A et al., (1995) Acta Orthop. Scand (S266) 66: 103-106). Investigations in several laboratories could not confirm the usefulness in measuring serum KS in arthritic patients.
Thrombospondin (450 kDa) is a high molecular weight adhesive glycoprotein consisting of three identical monomers of molecular weight 150 kDa. It is found in the cartilage and is produced by articular chondrocytes (Miller and McDevitt (1988), Biochem. Biophys. Res. Comm. 153: 708-714). To date there are no reports which document the presence of thrombospondin or its degradation products in the synovial fluid of arthritic patients.
Quantification of serum COMP may have prognostic value for rheumatoid arthritis and osteoarthritis (M Sharif, Saxne T, Shepstone L, Kirwan J R, Elson C J, Heinegard D, Dieppe P A: Br J Rheumatol 34: 4, 306-10,1995.; Hansson B M, Carey D, Alini M, lonescu M, Rosenberg L C, Poole A R, Heinegard D, Saxne T, J Clin Invest 95:1071-7, 1995). However, previous assays which quantify serum COMP do not allow determination of whether cartilage degradation is on-going in an arthritic patient.
Antigenic KS is present in elevated amounts in synovial fluid from human osteoarthritis joints (Shimozuru et al., Orthop. Trans. 20:419 1995). The levels of this and other markers of proteoglycan catabolism are highest during the pre-radiological stages of the disease and tend to drop with time, especially in joints exhibiting secondary inflammatory changes or loss of articular cartilage mass (see Thonar E J-M A et al., Sports medicine and arthroscopy review: chondral injuries (Ed. Andrish J. T). Raven Press, New York 1994: 13-29, for review). This makes interpretation of the data difficult. However, this difficulty can be circumvented by measuring additional markers and reporting the results as ratios of one marker to another (Thonar E J-M A et al., Acta Orthop. Scand. (Suppl 266) 66:103-106 1995). Interestingly, a recent report has claimed that the ratio of antigenic COMP to antigenic KS in the synovial fluid of the same patient may be useful in monitoring changes in cartilage macromolecue turnover (Peterson et al., (1997) Ann. Rheum. Disease 56: 64-67.)
Identification of new proteins or fragments of proteins (degradation products) or ratios of the different proteins or fragments whose presence in the synovial fluid and/or blood serum can be correlated with a disease state such as arthritis (osteoarthritis or rheumatoid arthritis) would be useful for diagnosis and treatment of such diseases. Moreover, the development of new antibodies and other molecules to detect such new proteins or fragments of proteins would allow easily utilized assays to be routinely applied for the diagnosis and treatment of patients with arthritis. For new drug development in arthritis there is a need to have assays to evaluate efficacy of the new drug on the cartilage matrix and a means to select patients for treatment therapies. The present invention has identified new low molecular weight fragments of the COMP protein that are correlated with the progression of arthritis, antibodies which bind to these fragments and assays to measure the severity of arthritis disease states by quantifying these fragments and other known COMP fragments and fragments of thrombospondin-1 (xe2x80x9cTSP-1xe2x80x9d). In our investigations using the anti-peptide antibody to the carboxy terminal of COMP, what was unforeseen is that specific breakdown products derived from the carboxyl terminal end of this molecule or a ratio of the breakdown products to KS would have diagnostic or prognostic value. It shows that the level of COMP is different than these C-terminal degradation products and reflect something different than the intact molecule. In addition, this antibody was found to recognize the other known COMP fragments. Moreover, this antipeptide antibody unexpectedly recognized the N-terminal 20 kDa fragment of bovine thrombospondin, and full-lenth human TSP-1. Since the N-terminal sequence of bovine and human thrombospondin are identical, this antibody is expected to recognize N-terminal 20 kDa fragment of human TSP-1.
The present invention is drawn to a group of novel and newly discovered low molecular weight protein fragments that are C-terminal degradation products of COMP. A first group, referred to herein as xe2x80x9cLMW-COMPxe2x80x9d fragments, have a molecular weight of about 14-33 kilodaltons on SDS-PAGE. Preferably, the LMW-COMP fragments have a molecular weight of about 30, 20, 18, 16 or 14 kilodaltons and most preferably the LMW-COMP is the predominant species of low molecular weight degradation products having an apparent molecular weight on SDS-PAGE electrophoresis of about 20 kilodaltons. This COMP fragment is produced in increasing amounts as the arthritis disease condition becomes more severe. These newly discovered C-terminal COMP fragments are only separated from other COMP components under reducing conditions and therefore their separation depends on assays wherein the separation of COMP fragments (if it occurs) is conducted under such reducing conditions. Another aspect of the present invention is polyclonal and monoclonal antibodies capable of binding to the new LMW-COMP fragments. The LMW-COMPs are recognized by antibodies, preferably monoclonal antibodies, to the amino acid polypeptide derived from at least the 17 amino acid carboxy terminus of COMP. These antibodies, upon Western blotting, detect another group of COMP C-terminal degradation fragments which have a molecular weight of from about 67 kDa to about 80 kDa and 150 kDa to 250 kDa and also cross-react with another arthritic degradation product which is a 20 kDa fragment of thrombospondin derived from the N-terminus of TSP-1. The LMW-COMP fragments, the 67-80 kDa and 220-250 kDa COMP fragments and the thrombospondin fragments are collectively referred to herein as xe2x80x9carthritic degradation productsxe2x80x9d or xe2x80x9cADPxe2x80x9d. Therefore the use of the term xe2x80x9carthritic degradation productsxe2x80x9d or xe2x80x9cADPxe2x80x9d can refer to all three fragments described above together or one or more of the fragments alone. The antibodies which recognize and bind to these fragments are, therefore, referred to as xe2x80x9cADP antibodiesxe2x80x9d. Still another aspect of the present invention is assay techniques to measure the level of ADP in various bodily fluids, preferably synovial fluid and serum.
A preferred embodiment of the assay aspect of the invention is an assay to measure the level of ADP comprising:
(1) incubating a body fluid sample, which has been obtained from an arthritis patient, under reducing or non-reducing conditions;
(2) incubating the body fluid sample with an anti-ADP antibody; and
(3) measuring the level of bound anti-ADP antibody in the body fluid sample.
In a most preferred embodiment of the assay, the ADP are LMW-COMP fragments.
Optionally, the level of bound anti-ADP antibody in the body fluid sample can be compared to the level of bound anti-ADP antibody in the body fluid sample at a different time during diagnosis and/or treatment of the arthritis.
Yet another aspect of the present invention is directed to diagnostic assays for measuring the ratio of the arthritic degradation products to the level of KS in a bodily fluid sample derived from a patient, preferably synovial fluid, and serum samples, for diagnosis and/or treatment of arthritis.